Structural model of the collagen-like region of C1q comprising the kink region and the fibre-like packing of the six triple helices

A detailed three-dimensional model of the collagenous part of C1q was derived by model building and computer-aided energy refinement calculations. The proposed structure is based on the collagen-like (-Gly-Xaa-Yaa-) repeating sequence of 78 to 81 residues in the N-terminal regions of the constituent A, B and C chains, on the mode of disulphide linkage between the 18 chains of C1q, and on its electron microscopically derived gross structure. It is demonstrated that the interruptions of the repeating sequence about half-way along the length of the collagenous regions (Gly36-Ile37-Arg38-Thr39 in the A chain and Ala36-Ile37-Hy138 in the C chain) do not lead to a disruption of the triple helical conformation but rather to a bend of about 60 degrees in an otherwise continuous triple helix. These features are consistent with a flexibility comparable with that of regular triple helices and with the observed low proteolytic susceptibility of the kink region. The azimuthal orientation of the kink is defined approximately by ArgA38 being located in the cap of the knee. Because of this extra residue between two glycine residues, a bad contact that would arise between the methyl group of AlaC36 and the peptide carbonyl of IleA37 in a straight triple helix is relaxed. The model features also a cluster of hydrophobic contacts between large hydrophobic side-chains in the interaction edges between the six collagen triple helices aligned with their about 10 nm long N-terminal regions in the fibril-like endpiece of C1q. The azimuthal orientations of the triple helices were derived by energy calculations of side-chain interactions previously applied to fibre-forming collagens. Independently, the same orientations and interaction edges were derived from the azimuthal orientation of the kink and the electron microscopically observed orientations of the triple helical arms that emerge from the endpiece, and which carry the C-terminal globular binding domains. The structural model has a number of implications for the assembly of the first component of complement from C1q and the zymogen complex C1r2C1s2 and possible mechanisms of its activation.     

Characterization of the binding sites for nimodipine and (-)-desmethoxyverapamil in bovine cardiac sarcolemma

The bovine cardiac sarcolemmal binding sites for the dihydropyridine nimodipine and the phenylalkylamine (-)-desmethoxyverapamil were studied. The density of the nimodipine and (-)-desmethoxyverapamil binding sites increased 8.3-fold and 3.4-fold with the sarcolemma. The binding sites for both compounds were destroyed by trypsin. Nimodipine bound in the presence of 1 mM free calcium to a high-affinity and a low-affinity site with apparent Kd values of 0.35 +/- 0.09 nM (n = 9) and 33 +/- 6.0 nM (n = 9) and with apparent densities of 0.3 +/- 0.05 pmol/mg (n = 9) and 8.2 +/- 1.0 pmol/mg (n = 9). The binding to the high-affinity site was abolished by 1 mM EGTA. The binding sites were specific for dihydropyridines. The (-)-isomers of several phenylalkylamines inhibited nimodipine binding by an apparent allosteric mechanism. (-)-Desmethoxyverapamil bound in the presence of 5 mM EGTA to a high-affinity and a low-affinity site with apparent Kd values of 1.4 +/- 0.3 nM (n = 6) and 171 +/- 26 nM (n = 6) and with apparent densities of 0.16 +/- 0.02 pmol/mg (n = 6) and 13.6 +/- 2.7 pmol/mg (n = 6). The binding to both sites was inhibited by calcium with a half-maximal concentration of 4.3 mM. The binding sites were specific for the other phenylalkylamines and had a higher affinity for the (-)-isomers than for the (+)-isomers. Nimodipine inhibited the binding of (-)-desmethoxyverapamil by an apparent allosteric mechanism. d-cis-Diltiazem inhibited non-competitively the binding of (-)-[3H]desmethoxyverapamil with a Ki of 3.7 microM. Diltiazem up to concentrations of 10 microM did not affect the amount of nimodipine bound at equilibrium at 20 degrees C. However, but in agreement with this result, diltiazem decreased threefold at 20 degrees C the dissociation and association rates for the high-affinity nimodipine receptor. These rates were only marginally affected at 4 degrees C and 37 degrees C. d-cis-Diltiazem reversed in a competitive manner the inhibition of nimodipine binding elicited by the addition of (-)-desmethoxyverapamil with a Ka value of 1.6 microM. The amount of nimodipine bound was inhibited by 50% by the adenosine uptake inhibitors nitrobenzylthioinosine and hexobendine with apparent median inhibitory concentrations of 1 nM and 3 nM, respectively. Nitrobenzylthioinosine completely abolished binding of nimodipine to the low-affinity site, but did not affect binding to the high-affinity site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Aerobic spore-forming bacteria as detrimental infectants in plant tissue cultures

Summary Aerobic spore-forming bacteria were isolated from plant tissue cultures from a commercial plant cultivation station. Bacillus circulans was found to be a detrimental infectant as a serious consequence of the heat-resistance of the endospores of these bacteria. They were extremely motile, utilized several growth-promoting factors, and could be eliminated by early microscopical identification, killing by heat treatment, or by using antibiotics or disinfecants.Dedicated to Professor Dr. Gustav Kortüm on the occasion of his 80th birthday     

Embryonic development and molting of the antennal coeloconic no pore- and double-walled wall pore sensilla in Locusta migratoria (Insecta,Orthopteroidea)

Summary The embryonic development of antennal coeloconic sensilla was studied at four stages between 132 and 252 h after oviposition in Locusta migratoria. Initially the anlagen of the sensilla consist of 2–4 sensory cells and 3 enveloping cells. Two additional cells contribute later to the formation of socket and pit. The dendritic outer segments of the sensory cells elongate before the trichogen process grows out (ecdysis type I) with exception of one sensory cell in anlagen of poreless (np) sensilla. Other differences between np and double-walled wall pore (dw wp) sensilla are not visible until at least about 220 h after oviposition. Molting, which was studied in four stages, follows ecdysis type I in both sensillum types. The fourth enveloping cell maintains its tight connection to the socket of the sensillum even after apolysis. Its apical portion is torn off and shed together with the old cuticle. The electron-dense material between the dendritic sheath and the cuticular wall of the peg in np sensilla, which is regarded important for stimulus transmission, is not deposited during retraction of the trichogen cell. The concentric walls and spoke channels characteristic of dw wp sensilla result from deposition of cuticular material around wedge-shaped projections of the trichogen cell. The typical trilaminar 15 nm cuticulin layer is produced only on the ridges of these sensilla. The first cuticular lining of the spoke channels is only 7 nm thick and of a different structure. A flocculent material surrounds the outgrowing trichogen process. It is continuous with the filling of the spoke channels and can thus be considered as component of the stimulus-transmitting material in the functioning intermolt dw wp sensilla.     

Identification of two types of keratin polypeptides within the acidic cytokeratin subfamily I

Cytoskeletal filaments of the α-keratin type (cytokeratins) are a characteristic of epithelial cells. In diverse mammals (man, cow and rodents) these cytokeratins consist of a family of approximately 20 polypeptides, which may be divided into the more acidic (I) and the more basic (II) subfamilies. These two subfamilies show only limited amino acid sequence homology. In contrast, nucleic acid hybridization experiments and peptide maps have been interpreted to show that polypeptides of the same subfamily share extended sequence homology.We compare two polypeptides of the acidic cytokeratin subfamily, VIb (M_r 54,000) and VII (M_r 50,000), which are co-expressed in large amounts in bovine epidermal keratinocytes. These two epidermal keratins can be distinguished by specific antibodies and show different patterns of expression among several bovine tissues and cultured cells. In addition, they differ in the stability of their complexes with basic keratin polypeptides and in their tryptic peptide maps. The amino acid sequences deduced from the nucleotide sequences of complementary DNA clones containing the 3′ ends of the messenger RNAs for these keratins are compared with each other and with available amino acid sequences of human, murine and amphibian epidermal keratins. Bovine keratins VIb and VII share considerable sequence homology in the α-helical portion (68% residues identical) but lack significant homology in the extrahelical portion. Bovine keratin VIb shows, in its α-helical region, a pronounced sequence homology (88% identity) to the murine epidermal keratin of M_r 59,000. In addition, the non-helical carboxy-terminal regions of both proteins are glycinerich and contain a canonic sequence GGG^S_GYGG, which may be repeated several times. Moreover, their mRNAs present a highly conserved stretch of 236 nucleotides containing, in the murine sequence, the end of the coding and all of the non-coding region (81% identical nucleotides). Bovine keratin VII is considerably different from the murine M_r 59,000 keratin but is almost identical to the human cytokeratin number 14 of M_r 50,000, both in the α-helical and in the non-α-helical regions of the proteins, and the mRNAs of the human and the bovine keratins also display a high homology in their 3′ non-coding ends.The results show that in the same species keratins of the same subfamily can differ considerably, whereas equivalent keratin polypeptides of different species are readily identified by characteristic sequence homologies in the α-helical and the non-helical regions as well as in the 3′ non-coding portions of their mRNAs. Among the members of the acidic subfamily I of cytokeratin polypeptides that are co-expressed in bovine epidermis, at least two types can be distinguished by their carboxy-terminal sequences. One type is characterized by its abundance of glycine residues, a consensus GGG^S_GYGG heptapeptide sequence, which may be repeated several times, and an extended stretch of high RNA sequence homology in the 3′ non-coding part. The other type shows a predominance of serine and valine residues, a subterminal GGG^S_GYGG sequence (which has been maintained in Xenopus, cow and man) and also a high level of homology in the 3′ non-coding part of the mRNA. The data indicate that individual keratin type specificity overrides species diversity, both at the protein and the mRNA level. We discuss the evolutionary conservation and the tissue distribution of these two types of acidic keratin polypeptides as well as their possible biological functions.     

CO2 sensitive receptors on labial palps ofRhodogastria moths (Lepidoptera: Arctiidae): physiology,fine structure and central projection

Summary The tips of the labial palps ofRhodogastria possess a pit housing uniform sensilla (Fig. 1), histologically characterized by wall-pores and receptor cells with lamellated outer dendrites (Fig. 2). The receptor cell axons project to glomeruli in the deutocerebrum (cf. Fig. 3) which are not innervated by antennal receptors. From their histology as well as from their central projection these sense organs are identical with palpal pit organs of other Lepidoptera (Lee et al. 1985; Kent et al. 1986; Lee and Altner 1986).Physiologically, the palp-pit receptors respond uniformly; they are most excitable by stimulation with carbon dioxide (Fig. 6) while they exhibit relatively moderate responses to various odorants (Fig. 4). The responses to CO₂ (Fig. 7) show a steep dose-response characteristic. In ambient atmosphere (i.e., ca. 0.03% CO₂) the cells are in an excited condition already; the seeming spontaneous activity exhibited in air is decreased if the preparation is kept under N₂ or O₂ or CO₂-free air (Figs. 7, 10). There is hardly any adaptation of the responses to continuous or repeated stimulation (Fig. 8). Perhaps CO₂ sensitivity is correlated with sensilla characterized by both wall-pores and lamellated dendrites. Pilot tests indicate that CO₂ perception might be widespread in the Lepidoptera (cf. Fig. 12), but the biological significance remains obscure.     

Similarity of activation of yeast phosphofructokinase by AMP and fructose-2,6-bisphosphate

Phosphofructokinase from yeast is effectively activated by AMP and fructose-2,6-bisphosphate by increasing the affinity of the enzyme to fructose-6-phosphate and the maximum activity toward this substrate. The enzyme is activated by AMP and fructose-2, 6-bisphosphate both at high and at low concentrations of ATP. The half maximum stimulation concentrations of AMP and fructose-2, 6-bisphosphate are about 200 microM and 2 microM, respectively. At saturating concentrations of AMP and fructose-2, 6-bisphosphate similar maximum activities were observed in the dependence of enzyme activity on the concentrations of fructose-6-phosphate. The fructose-6-phosphate affinity is more enhanced by fructose-2, 6-bisphosphate than by AMP.